Oligomer extended insulin-Fc conjugates

Abstract

This invention is in the field of protein conjugates. More specifically the invention relates to oligomer extended insulins with covalently attached Fc monomer polypeptides, for use in the treatment of a metabolic disorder or condition, and to methods of producing such oligomer extended insulin-Fc conjugates. The invention also relates to novel Fc fragments, to intermediate products, and to the use of such intermediate products in processes for the synthesis of the oligomer extended insulin-Fc conjugates of the invention. Finally the invention provides pharmaceutical compositions comprising the oligomer extended insulin-Fc conjugates of the invention, and relates to the use of such compositions for the treatment or prevention of medical conditions relating to metabolic disorders or conditions.

The invention claimed is: 
     
       1. An oligomer extended insulin-Fc conjugate represented by Formula I′: 
       
         
           
           
               
               
           
         
         wherein 
         Ins represents an analogue of human insulin, wherein said insulin analogue is a two-chain insulin molecule comprising an A- and a B-chain; 
         OEF indicates a polar recombinant extension fused to the insulin A-chain C-terminus, or fused to the insulin B-chain N-terminus, wherein the extension comprises a combination of (i) and (ii):
 (i) one or more of the following amino acid residues: Ala (A), Asp (D), Glu (E), Gly (G), Pro (P), Gln (Q), Ser (S), and Thr (T); and 
 (ii) a lysine (K) residue, to render an [OEF-Ins] construct; 
 
         the [OEF-Ins] construct is connected to the trivalent linking group X-U(-Z) 2  via the epsilon amino group of a lysine (K) residue; 
         X represents a covalent bond, or a divalent linking group, connecting [OEF-Ins] and U; 
         U represents a central trivalent linking unit, connecting the two Z-[S-Fc′] groups to X; or, in case X represents a covalent bond, U is connecting the two Z-[S-Fc′] groups to the epsilon amino group of a lysine residue in the [OEF-Ins] construct; 
         Z is absent or represents a divalent linking group connecting the central linking unit (U) to a thiol moiety (S) of a monomer Fc polypeptide (Fc′), or a fragment thereof; or, in case Z is absent, U is connected directly to [S-Fc′]; and 
         [S-Fc′] represents an Fc′ component, wherein the Fc′ component is a monomer Fc polypeptide or a fragment thereof, with the S representing a sulphur atom originating from a cysteine (C) residue comprised within the Fc′ component. 
       
     
     
       2. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein X represents
 a covalent bond; 
 a divalent linker of the structure —C(O)—(CH 2 ) 1 —C(O)—, wherein 1 represents an integer in the range of from 1 to 20; or 
 a divalent linker of the structure —C(O)—(CH 2 ) o —NH—C(O)—, wherein o represents an integer in the range of from 1 to 20. 
 
     
     
       3. The oligomer extended insulin-Fc conjugate according to  claim 1 , wherein the central trivalent linking unit (U) and divalent linking group (Z) are represented by Formula IV′ 
       
         
           
           
               
               
           
         
         wherein, 
         N is a nitrogen atom and represents the central linking unit (U) according to Formula I′; 
         L 1 -NH(CO)-L 3  and L 2 -NH(CO)-L 4 , respectively, represents the divalent linking group (Z) according to Formula I′; 
         * indicates the point of attachment to a sulphur atom originating from a disulphide bridge of Fc (i.e. the [S-Fc′] moiety according to Formula I′); 
         # indicates the point of attachment to X; and 
         L 1  and L 2 , independently of each other, represent —(CH 2 ) m1 —; wherein 
         m1 represents an integer in the range of from 1 to 6; and 
         L 3  and L 4 , independently of each other, represent —(CH 2 ) n1 —; 
         wherein n1 represents an integer in the range of from 1 to 6. 
       
     
     
       4. The oligomer extended insulin-Fc conjugate according to  claim 1 , wherein X represents a covalent bond and wherein U—(—Z) 2  represents a 3,5-disubstituted benzoyl moiety as illustrated by Formula VII: 
       
         
           
           
               
               
           
         
         wherein 
         indicates the point of attachment to the sulphur atom of Formula I′; 
         # indicates the point of attachment to the epsilon amino group in a lysine residue in the [OEF-Ins] construct; and 
         L 9  and L 10 , independently of each other, represents —(CH 2 ) n3 —; wherein 
         n3 represents an integer in the range of from 1 to 6. 
       
     
     
       5. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein
 Ins represents an analogue of human insulin containing one or more of the following substitutions: A 14A or A 14E, A21G or A21Q, B3Q, B16E or B16H, B25H, B29R, and desB30. 
 
     
     
       6. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein
 OEF represents a polar recombinant OEF extension fused to the insulin A-chain C-terminus, or fused to the insulin B-chain N-terminus, which extension includes one or more of the following amino acid residues: Ala (A), Glu (E), Gly (G), Pro (P), and Gln (Q), in a combination with a lysine (K) residue, to render an [OEF-Ins] construct with a total number of amino acid residues of the OEF in the range of 15 to 110. 
 
     
     
       7. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein Fc′ represents a monomer of an IgG1 Fc polypeptide, of an IgG2 Fc polypeptide, of an IgG4 Fc polypeptide, or a fragment thereof. 
     
     
       8. The oligomer extended insulin-Fc conjugate of  claim 1 , selected from the group consisting of
 (A14E, A21G, A22(GQEP) 19 , A98KK{circumflex over ( )}, A99P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 19 , A98KK{circumflex over ( )}, A99P, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, B(−78K{circumflex over ( )}), B(−77P), B(−1)(GQAPGQAPGQEP) 6 -GQAP, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, B(−78K{circumflex over ( )}), B(−77P), B(−1)(GQEP) 19 , B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQAPGQEP) 6 -GQAP, A98KK{circumflex over ( )}, A99P, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 19 , A98K{circumflex over ( )}, A99P, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 6 , A46K{circumflex over ( )}, A47P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 12 , A70K{circumflex over ( )}, A71P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQEP) 6 , A70K{circumflex over ( )}, A71P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 3 , A34G, A35Q, A36E, A37K{circumflex over ( )}, A38P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, B(−78K{circumflex over ( )}), B(−77P), B(−1)(GQEP) 19 , B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQEP) 19 , A98K{circumflex over ( )}, A99P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQAPGQEP) 6 -GQAP, A98K{circumflex over ( )}, A99P, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQEP) 6 , A70K{circumflex over ( )}, A71P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQAP) 19 , A98K{circumflex over ( )}, A99P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQAP) 19 , A98K{circumflex over ( )}, A99P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))6-[bis[2-[(2-acetyl)amino]ethyl]amino]-6-oxo-hexanoyl conjugate; 
 A14E, A21G, A22(GQEP) 12 , A70K{circumflex over ( )}, A71P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, B(−78K{circumflex over ( )}), B(−77P), B(−1)(GQAP) 19 , B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQAPGQEP) 6 -GQAP, A98K{circumflex over ( )}, A99P, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQAPGQAPGQEP) 6 -GQAP, A122K{circumflex over ( )}, A123P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21Q, A22(G) 18 , A40K{circumflex over ( )}, B25H, B29R, desB30 human insulin/(227A, 234A, 235K hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQAP) 24 , A118G, A119Q, A120A, A121K{circumflex over ( )}, A122P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))3,5-bis[(2-acetyl)amino]benzoyl conjugate; 
 (A14E, A21G, A22(GQAP) 24 , A118G, A119Q, A120A, A121K{circumflex over ( )}, A122P, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQAPGQAPGQEP) 6 -GQAP, A98K{circumflex over ( )}, A99P, B3Q, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]carbamoylamino]butanoyl conjugate; 
 (A14A, A21G, A22(GQAP) 18 , A94G, A95Q, A96A, A97K{circumflex over ( )}, A98P, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQAP) 18 , A94G, A95Q, A96A, A97K{circumflex over ( )}, A98P, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A1(N(alpha)acetyl), A14E, A21G, A22(GQAP) 19 , A98K{circumflex over ( )}, A99P, B1(N(alpha)acetyl), B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A21G, A22(GQAP) 18 , A94G, A95Q, A96A, A97K{circumflex over ( )}, A98P, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 (A14E, A21G, A22(GQAP) 24 , A118G, A119Q, A120A, A121K{circumflex over ( )}, A122P, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))3,5-bis[(2-acetyl)amino]benzoyl conjugate; 
 A14E, A21G, A22(GQAP) 18 , A94G, A95Q, A96A, A97K{circumflex over ( )}, A98P, B16E, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))3,5-bis[(2-acetyl)amino]benzoyl conjugate; 
 (A14E, A21G, A22(GQAP) 18 , A94G, A95Q, A96A, A97K{circumflex over ( )}, A98P, B16E, B25H, B29R, desB30 human insulin)/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; 
 A14E, A21G, A22(GQEP) 6 , A46K{circumflex over ( )}, A47P, B25H, B29R, desB30 human insulin/(227A, 234A, 235K, 297E, 315Q, 384Q, des447 hIgG4-Fc(228-447))4-[bis[2-[(2-acetyl)amino]ethyl]amino]-4-oxo-butanoyl conjugate; and 
 (A14E, A21G, A22(GQEP) 19 , 98K{circumflex over ( )}, A99P, B25H, B29R, desB30 human insulin)/(226A, 227G, 228P, 234A, 235K, des447 hIgG4-Fc(228-447))3,5-bis[(2-acetyl)amino]benzoyl conjugate. 
 
     
     
       9. A pharmaceutical composition comprising the insulin-Fc conjugate of  claim 1  and one or more pharmaceutically acceptable carriers or diluents. 
     
     
       10. A method of treatment or alleviation of a metabolic disease or disorder or condition of a living animal body, including a human, the method comprising administering to such a living animal body in need thereof a therapeutically effective amount of the oligomer extended insulin-Fc conjugate of  claim 1 . 
     
     
       11. The method of  claim 10 , wherein the metabolic disease or disorder is Type 1 diabetes or Type 2 diabetes. 
     
     
       12. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein the Fc′ component is aglycosylated. 
     
     
       13. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein the Fc′ component is a fragment of a hIgG4 derived Fc sequence, starting at position 228 and ending at position 447, which sequence contains one or more extensions and/or substitutions selected from the group of 226A or 226G, 227A or 227G, 228P, 234A, 235A or 235K, N297E or N297Q, 315Q, 384Q, and des447. 
     
     
       14. The oligomer extended insulin-Fc conjugate of  claim 13 , wherein the Fc′ component is 226A, 227G, 228P, 234A, 235K, des447 hIgG4-Fc(228-447). 
     
     
       15. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein OEF indicates a polar recombinant extension fused to the insulin A-chain C-terminus. 
     
     
       16. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein OEF indicates a polar recombinant extension fused to the insulin B-chain N-terminus. 
     
     
       17. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein X represents a covalent bond. 
     
     
       18. The oligomer extended insulin-Fc conjugate of  claim 1 , wherein the [OEF-Ins] construct is connected to the trivalent linking group X-U(-Z) 2  via the epsilon amino group of the K residue of the extension. 
     
     
       19. A method of lowering blood glucose in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the oligomer extended insulin-Fc conjugate of  claim 1 .